Detecting Waning Serological Response with Commercial Immunoassays: 18-Month Longitudinal Follow-up of Anti-SARS-CoV-2 Nucleocapsid Antibodies

ABSTRACT Past severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an important determinant of protection from reinfection and of postvaccine immune responses. Herein, we conducted a follow-up analysis of health care workers previously infected with coronavirus disease 2019 (COVID-19) with the aim of evaluating different immunoassays for their capability in detecting the waning anti-SARS-CoV-2 immune responses and accuracy in documenting past SARS-CoV-2 infections. We evaluated serum antinucleocapsid antibody levels in convalescent individuals following a 1.5-year interval from SARS-CoV-2 infection. Three different commercial immunoassays that qualitatively measure serum antibodies targeting the SARS-CoV-2 nucleocapsid protein, namely, the Abbott Architect SARS-CoV-2 IgG, the Euroimmun anti-SARS-CoV-2 NCP enzyme-linked immunosorbent assay (ELISA) IgG, and the Roche Elecsys anti-SARS-CoV-2, were tested for comparison of detectability. A total of 38 individuals consented to participating in this follow-up analysis. From assay to assay, seropositivity rate at 18 months from infection varied from lowest at 42% to highest at 92%. The Roche Elecsys immunoassay, dependent on the dual-antigen antibody detection method and tuned for the detection of high avidity antibodies, was most capable of accurately documenting past SARS-CoV-2 infections. Different immunoassays showed variable capability of determining previous infection status under waning antibody concentrations. Immunoassays with lower detection limits are to be selected, and adjusted thresholds are to be considered in order to maximize the tests’ performance. IMPORTANCE Past SARS-CoV-2 infection is an important determinant of protection from reinfection and of postvaccine immune responses. Our results show that different immunoassays, by design, harbor variable capability of tracking SARS-CoV-2 infection under waning antibody concentrations. With each recovered patient standing at a unique time point along the decline curve of antibodies, precise estimation of COVID-19 cumulative incidence remains a challenge. Since future surveillance studies will be targeting more than ever heterogenous cohorts, selecting the appropriate immunoassay is crucial in order to assure reliable decisions about an individual’s previous infection status.

to 18 months post-infection showing the difference in titers as a reflection of assay performance at this time point. As we are continuing to learn about the immunity to SARS-CoV-2 from primary infections and vaccines this observation is important to the scientific community. Major comments: Recent studies have shown that IgG levels can be impacted by the type of infection the patient originally experienced (symptomatic vs. asymptomatic) (Huynen et al 2022, Gerhards et al 2021. Huynen et al. 2022 showed IgG levels that were higher and increased over time in symptomatic subjects but were lower and stable in asymptomatic subjects. Can the authors comment on whether there was any impact on the IgG levels in the participants of this study based on the earlier infection and symptoms?
Line 240 and 247: Should state 18-months Minor Comments: Line 78: Define COVID-19 here as opposed to line 84. Line 92: Add manufacturer information for each immunoassay Line 94: How do the adjusted thresholds compare to the instructions for use from the manufacturer, please address. Line 113: Address why the titers for these patients may have increased. Line 127: Remove the redundant sentence "The pandemic having..." and consider revising it in line 61. Line 135: Consider adding the sentence: "Our results..." to the Importance section as it highlights an important aspect of this Observation.

Reviewer #2 (Comments for the Author):
This study compared the ability of three different high throughput assays to detect anti-nucleocapsid antibodies 18 months after initial infection. They show notable differences in positivity rates for the Roche assay as compared to Abbott and Euroimmun. Comments for the authors: 1. Indicate that these are qualitative assays against the nucleocapsid antigen in the abstract and methods section. 2. Clarify that there really are no policies in place that currently take into account serologic testing as a means to determine the need for additional boosters. 3. One of the key limitations here is the limited number of individuals included in this long term study. 4. Can the authors comment on how their findings should be used to guide the public health response, if at all? This would circle back to their initial comments on use of serology to help potentially guide vaccination needs in the future.

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If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website. We honestly thank the reviewer for the very interesting comment and perfectly agree that the severity of COVID-19 is one of the strongest determinants of an individual's immune response, and may as well have an effect on future seroreversion rates.
Along with the works by Huynen and Gerhards, our group has also previously stated in an article already published in your journal that pauci-/asymptomatic COVID-19 cases are associated with attenuated immune responses (Nakagama Y, et al. Microbiol Spectr. 2021 Oct 31;9(2):e0108221. doi: 10.1128/Spectrum.01082-21). Combining the evidence, symptomatic cases are likely to elicit a more robust, as well as a longer-lasting, immune responses compared with asymptomatic infections. In the present study, however, we were too underpowered to assess for any correlation between COVID-19 severity and seroreversion rates. We have listed this among the Limitations and stated the need for a larger-scaled analysis to efficiently approach this hypothesis.
page 10, lines 233-241 A reference has been added.
page 15, lines 365-368 A further interesting aspect regarding your comment was the possible contrast between severity group in the kinetics of antibody response. We have clearly shown in a study published in your sister journal (Ref #12, Nakagama Y, et al. 2022. A dual-antigen SARS-CoV-2 serological assay reflects antibody avidity. J Clin Microbiol 60:e0226221.) that the immunoassays' design is another major determinant of antibody kinetics.
Immunoassays, each to different extent, reflect the maturity and avidity of the serum antibodies. The design of the Roche Elecsys immunoassay, namely the dual-antigen binding method, makes the assay less likely to capture low-affinity antibodies that are predominant during the early stage of convalescence. Thus, when assessing sera from early-convalescence, the different immunoassays' variable likelihood of detecting such low-affinity antibodies has larger impact on measured antibody level, and subsequently lead to poorer inter-assay agreement. In later-convalescence, the sera become rich in higher-affinity antibodies which are more Firstly, […]. Being underpowered in design has also hindered the study from assessing for any predictors of seroreversion. However, as we and others have previously described, COVID-19 severity is among the strongest determinants of an individual's serological response (3,16). Larger scaled analyses are warranted to further explore the association between symptom severity and seroreversion rates.  We honestly thank the reviewer for the correction, and apologize about the erroneous description in the initial submission. The correction appears now, as below.
We have recently reported that immunoassays […] the analyte serum antibodiesthe uniqueness in design of the Roche Elecsys immunoassay, namely the dual-antigen binding method, makes the assay less likely to capture low-affinity antibodies that are predominant during the early stage of convalescence (12). The design of […] the receiver operating characteristic curve analysisThus, when assessing sera from early-convalescence, the different immunoassays' variable likelihood of detecting such low-affinity antibodies may have larger impact on the measured antibody level, and subsequently lead to poorer inter-assay agreement. In later-convalescence, the affinity maturation of antibodies progresses and the sera become rich in higher-affinity antibodies secreted by the long-lived plasma cells. These higher-affinity antibodies are more readily detected with the dual-antigen binding method. Therefore, when measured with the Roche Elecsys immunoassay, an individual with highly efficient maturation in the affinity of antibodies may experience an increase in relative antibody levels over time (12) Thank you, reviewer, for the correction. We also apologize about the erroneous description in the initial submission. The correction appears now, as below.

Comment 4;
Line 92: Add manufacturer information for each immunoassay We thank the reviewer for the comment. We have added some information to the original manuscript. I believe that the information below, correctly specifies the  Line 113: Address why the titers for these patients may have increased.
We thank the reviewer for the comment and noticed that we have fell short of sufficiently explaining the mechanism.
As stated in our 'Response to Comment #1', the design of the Roche Elecsys immunoassay, namely the dual-antigen binding method, makes the assay less likely to We thank the reviewer for the comment, and apologize for our redundancy.
The sentence has now been discarded, and the context had it appeared is now somewhat rephrased, as below.
page 4, lines 65-69 The pandemic having prevailed for more than two years, With each recovered patient will now positioningstanding at a unique time point along the decline curve of antibodies, precise estimation of COVID-19 cumulative incidence remains a challenge. Thus, Since future surveillance studies will be targeting more than ever heterogenous cohorts, selecting the appropriate immunoassay is crucial in order to assure reliable decision on an individual's previous infection status. 13. National SARS-CoV-2 Serology Assay Evaluation Group. 2020. Conversely, a study has found a seroreversion (loss of antibodies) rate of over 30% at eight months of follow-up with the Abbott ARCHITECT anti-nucleocapsid immunoassay(10). The pandemic having prevailed for more than two years, each recovered patient will now position at a unique time point along their decline curve of antibodies.
[…] Since past COVID-19 infection is a strong predictor of protection from symptomatic reinfection and of post-vaccine immune responses, it is considerably applicable to prioritize the distribution of primary and further booster vaccinations based on anti-nucleocapsid antibody serology (11,12). Our results, with one of the longest-term follow-up data, further build on the growing evidence that the interpretation of serological kinetics against SARS-CoV-2 are dependent on assay platform, and further stress the importance of selecting the appropriate assay that will assure reliable results(11).
Our results show that different immunoassays, by design, harbor variable capability of tracking SARS-CoV-2 infection under waning antibody concentrations.

Responses to comments made by Reviewer #2:
This study compared the ability of three different high throughput assays to detect anti-nucleocapsid antibodies 18 months after initial infection. They show notable differences in positivity rates for the Roche assay as compared to Abbott and Euroimmun.

Comment 1;
Indicate that these are qualitative assays against the nucleocapsid antigen in the abstract and methods section.
We appreciate the comment. The qualitative nature of the assays are now clearly indicated in the Abstract and the Methods sections. Taking this comment into consideration, we have replaced the term "titer", which gives the impression of a strictly 'quantitative' measure, with the word "level", wherever appropriate throughout the text.
Every substitution of the term is now highlighted in red font using the Word track-changes function. Clarify that there really are no policies in place that currently take into account serologic testing as a means to determine the need for additional boosters.
We fully appreciate the reviewer's comment. In fact, it is absolutely true that no policies currently consider an individual's prior infection status nor serology in the Serum samples were tested for antibodies targeting the SARS-CoV-2 nucleocapsid protein using three different commercial immunoassays which qualitatively measure serum antibodies targeting the SARS-CoV-2 nucleocapsid protein.
Three different commercial immunoassays which qualitatively measure serum antibodies targeting the SARS-CoV-2 nucleocapsid protein, namely […], were tested for comparison of detectability.
designing of vaccination strategies. Considering the still controversial issues regarding the future needs of additional boosters, as well as their optimal timing and dose, in previously infected individuals, we modified the text so as to avoid declarative phrasings.
We have modified the discussions as below.

One of the key limitations here is the limited number of individuals included in this long term study.
We fully appreciate the reviewer's comment. We have now clearly mentioned this in the Limitations section.
Anti-nucleocapsid serology, assessed in parallel with the neutralizing antibody responses, shall guide us to improved understanding of our hybrid immunity against SARS-CoV-2. Indeed, no policies currently consider an individual's prior infection status nor serology in the designing of vaccination strategies. However, faced with the increasing evidence that vaccinees with a previous SARS-CoV-2 infection are better protected from the virus than those without (13,14), the additional boosters' risk-benefit profiles for the previously infected individuals, as well as their optimal timing and dose, shall stay a matter of continuing interest. We appreciate the reviewer's comment. The incremental effect of prior COVID-19 infection on post-vaccine immune protection (hybrid immunity) has been increasingly evaluated, as stated in the WHO's interim statement (https://www.who.int/news/item/01-06-2022-interim-statement-on-hybrid-immunity-an d-increasing-population-seroprevalence-rates). For those with prior infection, the decay in antibody titer following a set of SARS-CoV-2 vaccination was far more gradual, leading to an approximate 4-fold change in the long-term antibody titer (Havervall S, et al. Clin Transl Immunology. 2022 Apr 18;11(4):e1388. doi: 10.1002/cti2.1388.
We believe that, with more evidence, integrating infection and vaccination-induced immunity into vaccination strategies and/or schedules may provide gains through the optimization of immunization schedules tailored to each country/community with different levels of community transmission. Likewise, the additional boosters' risk-benefit profiles for the previously infected individuals, as well as their optimal timing and dose, shall stay a matter of continuing interest. The increasing evidence from the literature have now been newly cited in the manuscript, as The present study has targeted a limited number of individuals: healthcare workers mostly generally of younger age with few comorbidities, resulting overall in rather mild COVID-19 phenotypes; only 1/38 required oxygen supplementation. The immune response may not be representative of the overall population may differ in kinetics and intensity if the analyzed cohort are to include cases of greater severity.
below, in order to back up our statement.
page 5, lines 87-92 page 9, lines 209-232 The reference list has been updated, according to their appearance in the text.
page 12, lines 282-286 Past SARS-CoV-2 […] (1). Individuals with a previous SARS-CoV-2 infection not only enjoy longer-lasting post-vaccination antibody levels, but also are known to achieve more efficient protection from clinical re-infection(2). For those with pauci-/asymptomatic infections who, for various reasons, did not receive an acute diagnosis, its later confirmation is only possible serologically(3,4). Therefore, robust serological assays are increasingly needed that assure reliable results despite of the waning antibody responses. Thus, anti-nucleocapsid antibody decay becomes a special concern when policy makers are seeking for a reliable index (e.g. past SARS-CoV-2 infection) upon which variations in their immunization programs can be made, in order to prioritize the immunization of more vulnerable populations (2).
Anti-nucleocapsid serology, assessed in parallel with the neutralizing antibody responses, shall guide us to improved understanding of our hybrid immunity against SARS-CoV-2. Indeed, no policies currently consider an individual's prior infection status nor serology in the designing of vaccination strategies. However, faced with the increasing evidence that vaccinees with a previous SARS-CoV-2 infection are better protected from the virus than those without (14,15), the additional boosters' risk-benefit profiles for the previously infected individuals, as well as their optimal timing and dose, shall stay a matter of continuing interest.